PROJECT SUMMARY This proposal builds upon exciting new paradigm defined by isolated human NK cell deficiency resulting from hypomorphic mutations in multiple structural components of the eukaryotic DNA helicase. The discovery of multiple patient cohorts with a strikingly similar NK cell phenotype, namely that of impaired terminal maturation leading to susceptibility to severe viral infection and malignancy, underscores the impact of these mutations on human NK cell development and function. Despite this compelling new insight driven by human disease, the requirement for the DNA helicase complex specifically in NK cell function has not been defined. In the proposed work, we will define this requirement in the context of NK cell differentiation and proliferation to make important advances in both NK cell biology and human health. Despite their documented requirement in determining the outcome and success of transplantation, NK cell development and acquisition of function is poorly understood. A powerful tool in defining requirements for human NK cell differentiation is the study of patients with monogenic causes of impaired NK cell maturation leading to isolated NK cell deficiency (NKD). Using this approach, we and others have identified novel NKD resulting from mutations in the CDC45-MCM-GINS DNA helicase complex and associated proteins. In the proposed work, we will define the requirement for the CMG complex and, more broadly, proliferation and cell division in human NK cell development. Specifically, we will 1) define the effect of CMG helicase mutations on peripheral blood subset heterogeneity using single cell RNA sequencing and unbiased quantitative flow cytometry. Furthermore, we have 2) developed a model for patient mutations that will allow us to dissect the effect of these mutations on NK cell differentiation from earliest precursor to mature cell. We will use this, combined with our validated in vitro NK cell differentiation system, to dissect the mechanism by which hypomorphic CMG mutations affect human NK cell development and acquisition of lytic function. Finally, we will 3) define the effect of CMG helicase mutations on the human NK cell antiviral response. Using careful analysis of gene expression, phenotype and function, we will determine the timing and the nature of developmental deregulation in NK cell developmental intermediates with hypomorphic replisome mutations. These aims we will advance a novel new paradigm in human NK cell differentiation, namely the specific requirement for proliferation and cell cycle control for NK cell terminal maturation. These studies have significant clinical importance for understanding the differentiation of NK cells in the unique milieu following hematopoietic stem cell transplant. Defining the role of proliferation in the generation of mature NK cell effectors will enable the better control of these cells to prevent graft vs. host disease and promote their natural anti-tumor immunity.